White rust resistant Chrysanthemum plants

ABSTRACT

Provided herein are white rust resistant plants of the genus  Chrysanthemum  and seeds, plant parts, plant cells and progeny thereof. Also provided herein are markers for identifying white rust resistant plants of the genus  Chrysanthemum . Specifically, the present invention relates to plant belonging to the genus  Chrysanthemum , the plants are resistant to white rust and the plants comprise in their genome at least one genomic region, or gene or allele, providing white rust resistance, the white rust resistance providing genomic region, or gene or allele, is genetically linked to a nucleic acid sequence comprised in at least one copy in the genome of the resistant plants and is represented by SEQ ID No. 2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States national phase of InternationalApplication No. PCT/EP2016/073671 filed Oct. 4, 2016, and claimspriority to Dutch Patent Application No. 2015575 and InternationalApplication No. PCT/EP2016/057327, filed Oct. 6, 2015, and Apr. 4, 2016,respectively, the disclosures of which are hereby incorporated in theirentirety by reference.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 1802675_ST25.txt. The size of thetext file is 1,033 bytes, and the text file was created on Mar. 22,2018.

The present invention relates to white rust resistant plants of thegenus Chrysanthemum and to seeds, plant parts, plant cells and progenythereof. The present invention further relates to means, andparticularly molecular markers, for identifying white rust resistantplants of the genus Chrysanthemum.

Chrysanthemums, also designated as chrysant(h)s, are flowering plants ofthe genus Chrysanthemum in the family Asteraceae. The plants are nativeto Asia and north-eastern Europe and are comprised of a large number ofhorticultural varieties and cultivars.

Several genera of Chrysanthemums amongst which the economicallyimportant florist's Chrysanthemums were classified in the genusDendranthema in the past. However, presently, the florist'sChrysanthemums are reclassified as Chrysanthemum indicum, restoring theposition of these Chrysanthemums in the genus Chrysanthemum.

Naturally occurring Chrysanthemum species are herbaceous perennialplants. These Chrysanthemum species display alternately arranged leavesdivided into leaflets with toothed or occasionally smooth edges.Chrysanthemums were first cultivated in China as a flowering herb as farback as the 15th century BC and over 500 cultivars had been recorded bythe year 1630.

Presently cultivated Chrysanthemums display a more pronounced andaesthetic flowering as compared to their wild relatives. The flowerheads occur in various forms, and can be daisy-like or decorative, likepompons or buttons. This genus contains many hybrids and thousands ofcultivars developed for horticultural purposes. In addition to thetraditional yellow, other colours are available, such as white, purple,and red. The most important hybrid is Chrysanthemum x morifolium, alsodesignated as Chrysanthemum x grandiflorum, being primarily derived fromChrysanthemum indicum.

Chrysanthemums can be divided into two basic groups, garden hardy andexhibition. Garden Chrysanthemums are perennials capable of wintering inmost northern latitudes. Exhibition varieties are generally not capableof surviving winter. Garden Chrysanthemums can be defined by theirability to produce an abundance of small blooms with little if anymechanical assistance, such as staking, and being able to withstand windand rain. Exhibition varieties generally require staking, overwinteringin a relatively dry, cool environment, and sometimes the addition ofnight lights.

White rust is a disease in plants caused by Basisiomycota. Basisiomycotaform a distinct phylogenetic lineage of fungus-like eukaryoticmicroorganisms. They are filamentous, microscopic, absorptive organismsthat reproduce both sexually and asexually. Basiodiomycetes occupy bothsaprophytic and pathogenic lifestyles, and include some of the mostnotorious pathogens of plants, causing devastating diseases such assouthern blight of potato, tomato and a wide range of ornamentals andstem rust of wheat. The basidiomycetes are best known for the productionof large fruit bodies such as mushrooms, puffballs and brackets and areimportant organisms in the decay of wood and leaf litter.

In Chrysanthemum, white rust is a disease generally caused by thepathogenic basidiomycete, or fungus, Puccinia horiana. Chrysanthemumspecific symptoms include white rust spots on the upper surfaces ofleaves. These spots are initially pale-green to yellow in colour and upto 5 mm in diameter, but may turn brown as the tissue becomes necrotic.On the underside of the leaf, the spots develop into pink or whitepustules that become prominent as the teliospores develop. The diseaseis generally carried on infected cuttings and plants, including cutflowers, of glasshouse Chrysanthemums.

Until 1963, Puccinia horiana was confined to China and Japan. However,it has since spread rapidly on infected imported cuttings and is now afeared and serious disease in nurseries in Europe. A large number ofpathotypes of P. horiana is known, and great differences of virulence ofpathotypes of P. horiana after inoculation on various Chrysanthemumcultivars was demonstrated (De Backer, 2012). The P. horiana pathotypeNL1, collected in 2006 in The Netherlands showed to be the most virulentone.

Preventive spraying with fungicides is effective but costly. When theclimate is very suitable for white rust even preventive sprays are noteffective enough and susceptible varieties are highly likely to beinfected. Active ingredients found useful include oxycarboxin,triforine, benodanil, triadimefon, diclobutrazol, bitertanol andpropiconazole. Verticillium lecanii has been suggested for biologicalcontrol of on glasshouse Chrysanthemums.

Considering the considerable damage to Chrysanthemum cultivation bywhite rust, there is a need in the art to provide new genetic resistantsources, i.e. there is a need in the art for new resistance genes oralleles providing durable white rust resistance to plants of the genusChrysanthemum.

It is an object of the present invention, amongst other objects, to meetthe above need of the art.

According to the present invention, this object is met by the presentinvention by providing plants, plant parts, seeds and means as outlinedin the appended claims.

Specifically, according to a first aspect, this object of the presentinvention, amongst other objects, is met by providing plants belongingto the genus Chrysanthemum, the plants are resistant to white rust andthe plants comprise in their genome at least one genomic region, or geneor allele, providing white rust resistance, the white rust resistanceproviding genomic region, or gene or allele, is genetically linked to anucleic acid sequence comprised in the genome of the resistant plantsrepresented by SEQ ID No. 2. Considering the hexaploid nature of plantsbelonging to the genus Chrysanthemum, SEQ ID No. 2 is preferably presentin the genome of the resistant plants in at least one copy, such as 2,3, 4, 5 or 6 copies per resistant plant.

Preferably, the present plants further comprise in their genome afurther genomic region, or gene or allele, providing white rustresistance, the further white rust resistance providing genomic region,or gene or allele, is genetically linked to a nucleic acid sequencecomprised in at least one copy the genome of the resistant plantsrepresented by SEQ ID No. 1. Considering the hexaploid nature of plantsbelonging to the genus Chrysanthemum, SEQ ID No. 1 is preferably presentin the genome of the resistant plants in at least two copies, such as 3,4, 5 or 6 copies.

The sequences of SEQ ID No. 1 and SEQ ID No. 2 are genetically linked towhite rust resistances providing genomic regions, also designated hereinas alleles or genes, or formulated differently, SEQ ID No 0.1 and SEQ IDNo. 2 are molecular markers indicative for the presence of white rustresistance genomic regions, alleles or genes. SEQ ID No. 1 and SEQ IDNo. 2 can, for example, be obtained by submitting a sample comprisinggenomic DNA of a white rust resistant plant of the genus Chrysanthemumto a restriction digestion with the restriction enzymes Mse1 and EcoR1optionally in combination a nucleic acid amplification using primerspairs developed based on the sequences provided herein.

Although detecting the presence of SEQ ID No. 1 and/or SEQ ID No. 2 issufficient to establish whether a plant of the genus Chrysanthemum isresistant to white rust, the resistance can additionally be confirmed bya disease assay such as the disease assay outlined below.

A disease assay can be conducted on cuttings or small plants insideclosed plastic containers 125 cm length×80 cm width×35 cm height using aplastic cover. White rust infected inoculum plants are placed in thecontainers (36 inoculum plants per aquarium evenly distributed among 265cuttings). An isolate derived from P. horiana pathotype NL1 was used;the original NL1 pathotype was obtained from the PlantenziektenkundigeDienst, Wageningen, NL. Because white rust requires a high relativehumidity and a water film on the leaves, the small plants or cuttings,the inner sides of the plastic container and the cover are preferablyfogged with demineralized water in combination with a wet cloth on thebottom of the plastic container. After preparing the experimental setup,the containers are placed in a growth chamber at 18° C. covered withwhite plastic to create a dark, humid environment for four days. Everyday the containers are ventilated to ensure a good dispersal of thespores through the whole aquarium. After removing the white plastic thecontainers are illuminated with a combination of mercury and SON-Tlights during 18 hours per day (6000 Lux). Generally, disease symptomscan be assessed 21 to 28 days post infection. Plants are scoredaccording to a scale of 1 (infection) to 9 (not infected) wherein ascores of 1-3 indicate the plant as being susceptible, 4-6 indicates theplant as being intermediate resistant and 7-9 indicates the plant asbeing resistant.

The present plants are preferably a cut Chrysanthemum plant or a potChrysanthemum plant. Cut Chrysanthemum plants and pot Chrysanthemumplants are also designated in the art as cut flowers and potted plants,respectively. Sometimes, also reference is made to garden Chrysanthemumsbeing a garden proof version of a pot Chrysanthemum plant.

According to a preferred embodiment of the invention, the present plantsbelonging to the genus Chrysanthemum are selected from the groupconsisting of Chrysanthemum x morifolium; Chrysanthemum x grandiflorum;Chrysanthemum x rubellum; Chrysanthemum abolinii; Chrysanthemumachillaea L.; Chrysanthemum alabasicum; Chrysanthemum brachyanthum;Chrysanthemum carinatum; Chrysanthemum chalchingolicum; Chrysanthemumcinerariifolium; Chrysanthemum coccineum; Chrysanthemum coreanum;Chrysanthemum coronarium; Chrysanthemum decaisneanum; Chrysanthemumdelavayanum; Chrysanthemum dichrum; Chrysanthemum fastigiatum;Chrysanthemum frutescens; Chrysanthemum gracile; Chrysanthemum grubovii;Chrysanthemum horaimontanum; Chrysanthemum hypoleucum; Chrysanthemumindicum L.; Chrysanthemum junnanicum; Chrysanthemum kinokuniense;Chrysanthemum kokanicum; Chrysanthemum konoanum; Crysanthemum majus;Chrysanthemum marginatum; Chrysanthemum mawei; Chrysanthemum maximum L.;Chrysanthemum miyatojimense; Chrysanthemum morifolium; Chrysanthemummultifidum; Chrysanthemum nitidum; Chrysanthemum parvifolium;Chrysanthemum przewalskii; Chrysanthemum purpureiflorum; Chrysanthemumramosum; Chrysanthemum rhombifolium; Chrysanthemum roborowskii;Chrysanthemum segetum; Chrysanthemum shihchuanum; Chrysanthemumshimotomaii; Chrysanthemum trilobatum; Chrysanthemum tripinnatisectum;Chrysanthemum vestitum; Chrysanthemum vulgare (L.); Chrysanthemumyoshinyanthemum; and Chrysanthemum zawadskii.

According to another preferred embodiment of the present invention, thecausative pathogen causing the present white rust disease is Pucciniahoriana. Puccinia horiana is an autoecious rust. The bicellularteliospores germinate in situ to produce unicellular basidiospores whichare dispersed in air currents. No other spores are known. High humidity,and a film of moisture, appear to be necessary for the germination ofboth teliospores and basidiospores. Teliospores are capable ofgermination as soon as they are mature; germination and discharge ofbasidiospores occur between 4° C. and 23° C. and, at an optimumtemperature of 17° C., discharge of basidiospores is observed within 3hours. Basidiospores can germinate over a wide temperature range and, at17-24° C., either surface of the leaf may be penetrated within 2 hours.Thus, only 5 hours of wetness is sufficient for a new infection tobecome established. Within the leaf, abundant, hyaline, intercellularhyphae are produced with intracellular haustoria. The incubation periodis normally 7-10 days, but short periods of high temperatures (over 30°C.) can prolong the period to 8 weeks.

Following infection, pale-green to yellow spots, up to 5 mm in diameter,develop on the upper surface of leaves. The centres of these spotsbecome brown and necrotic with aging. On the corresponding lowersurface, raised, buff or pinkish, waxy pustules (telia) are found. Asthe spots on the upper surface become sunken, these pustules becomequite prominent and turn whitish when basidiospores are produced. Teliaare occasionally found on the upper leaf surface. Severely attackedleaves wilt, hang down the stem and gradually dry up completely.

According to yet another preferred embodiment, the present inventionrelates to plants having a resistance to white rust substantiallysimilar to, or equivalent with, the resistance to white rust of aChrysanthemum plant deposited under number NCIMB 42455 on Mar. 14, 2016.These plants can be readily identified by comparing white rust diseaseresistance, for example using a disease assay as described hereinalthough other know white rust disease assays can be used. AChrysanthemum plant deposited under number NCIMB 42455 can be obtainedthrough the National Collection of Industrial, Food and Marine Bacteria(NCIMB), Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, United Kingdom.

According to a particularly preferred embodiment, the present plantshave a resistance to white rust identical to the white rust resistancefound in a Chrysanthemum plant deposited under number NCIMB 42455.Especially preferred are plants comprising at least one white rustresistance providing genomic region or gene, preferably at least twowhite rust resistance providing genomic regions or genes, derived from aChrysanthemum plant deposited under number NCIMB 42455 wherein thepresence of said white rust resistance providing genomic regions orgenes is indicated by the presence of SEQ ID No. 1 or SEQ ID No. 2 inthe genome of said plant. Formulated differently, SEQ ID No. 1 or SEQ IDNo. 2 are molecular, or genomic, markers indicative for, thusgenetically linked with, the present resistance providing genomicregions or genes which can, for example, be found in NCIMB 42455.

According to a yet another particularly preferred embodiment, thepresent plants comprise in their genome at least two white rustresistance providing genomic regions or genes, the first white rustresistance providing genomic region or gene is genetically linked to agenomic nucleic acid sequence comprised in said plant genome representedby SEQ ID No. 1 and the second white rust resistance providing genomicregion or gene is genetically linked to a genomic nucleic acid sequencecomprised in said plant genome represented by SEQ ID No. 2.

According to a most preferred embodiment, the present invention relatesto a Chrysanthemum x morifolium plant, wherein the plant is resistant tothe white rust pathogen Puccinia horiana and the white rust resistanceis encoded by a first resistance providing genomic region or genegenetically linked to SEQ ID No. 1 and/or a second resistance providinggenomic region or gene genetically linked to SEQ ID No. 2.

Considering the importance of white rust resistant genetic sources inthe art, such as the present plants, the present invention, according toa second aspect, relates to seeds, plant parts or plant cells of thepresent plants. The present seeds, plant parts or plant cells comprisein their genome SEQ ID No. 2 or SEQ ID No. 2 and SEQ ID No. 1,preferably SEQ ID No. 1 and SEQ ID No. 2 and are, accordingly, capableof providing, or cultivated into, plants being resistant to white rustand especially white rust caused by an infection with Puccinia horiana.

According to a third aspect, the present invention also relates toprogeny of the present Chrysanthemum plants. Progeny of the presentplants can be readily identified by establishing the presence of SEQ IDNo. 2, preferably SEQ ID No. 1 and SEQ ID No. 2, in their genome.

According to a fourth aspect, the present invention relates to the useof SEQ ID No. 1 or SEQ ID No. 2 for identifying a white rust resistantChrysanthemum plant. Suitable methods, based on SEQ ID No. 1 or SEQ IDNo. 2, for identifying such plant are generally known in the art such asmethods based on nucleic acid amplification of genomic DNA andsubsequent visualisation of amplification fragments although othertechniques can be envisaged such as techniques based on hybridisation.

The present invention will be further detailed in the example presentedbelow.

EXAMPLE Introduction

Martin, P., & Firman, I. (1970). Resistance of Chrysanthemum Cultivarsto White Rust (Puccinia horiana). Plant Pathology, 180-184 disclosesseveral varieties of Chrysantemum white rust resistant plants. In orderto asses whether the present genomic sequences linked white rust, i.e.SEQ ID Nos 1 and 2, are found in the disclosed chrysanthemum cultivars,these cultivars were subjected to marker analyses and the results arepresented in Table 1 below:

TABLE 1 Phenotype after inoculation with NL1 isolate of P. horiana ofChrysanthemum varieties previously reported by Martin (1970) to beresistant or immune to P. horiana. Phenotype according to Martin SEQ IDSEQ ID Variety (1970) Phenotype No. 1² No. 2 Alec Bedser Immune N.t. ¹ —— Fred Shoesmith Immune Susceptible — — Marlene Immune Susceptible — —Polaris Immune Susceptible — — Regalia Immune N.t. + — Streamer ImmuneSusceptible N.t. N.t. Sweetheart Immune Susceptible — — Target ImmuneN.t. — — Vibrant⁴ Immune N.t. N.t. N.t. Bravo Moderate Susceptible N.t.N.t. resistant Corsair Practically Susceptible — — immune DiscoveryPractically Susceptible — — immune Glamour Practically Resistant + —immune Rivalry Practically Susceptible — — immune ¹ N.t. = not tested²—: SEQ ID is not present, +: gene is presentAs can be clearly seen, none of the above plants disclosed in Martin etal., comprise a genomic sequence represented by SEQ ID No. 2.

The invention claimed is:
 1. A Chrysanthemum plant having resistance towhite rust and comprising in its genome SEQ ID NO: 2, seed of said planthaving been deposited under NCIMB Accession No.
 42455. 2. TheChrysanthemum plant of claim 1, wherein the Chrysanthemum plantcomprises in its genome SEQ ID NO:
 1. 3. The Chrysanthemum plant ofclaim 1, wherein a causative pathogen of said white rust is Pucciniahoriana.
 4. Progeny of the Chrysanthemum plant of claim 1, wherein theprogeny is resistant to white rust and comprises in its genome SEQ IDNO:
 2. 5. The Chrysanthemum plant of claim 4, wherein said plant is acut Chrysanthemum or said plant is a pot Chrysanthemum.
 6. TheChrysanthemum plant of claim 4, wherein said plant is a Chrysanthemum xmorifolium plant, said plant is resistant to the white rust pathogenPuccinia horiana, and said resistance is encoded by a first resistanceproviding genomic region, gene, or allele genetically linked to SEQ IDNO: 2 and a second resistance providing genomic region, gene, or allele,genetically linked to SEQ ID NO:
 1. 7. A seed, plant part, or plant cellof the Chrysanthemum plant of claim 1, wherein the seed, plant part, orplant cell is resistant to white rust and comprises in its genome SEQ IDNO:
 2. 8. A seed, plant part, or plant cell of the Chrysanthemum plantof claim 4, wherein the seed, plant part, or plant cell is resistant towhite rust and comprises in its genome SEQ ID NO:
 2. 9. A Chrysanthemumplant grown from seed deposited under NCIMB Accession No. 42455, whereinthe Chrysanthemum plant is resistant to white rust and comprises in itsgenome SEQ ID NO:
 2. 10. A seed, plant part, or plant cell of theChrysanthemum plant of claim 9, wherein the seed, plant part, or plantcell is resistant to white rust and comprises in its genome SEQ ID NO:2.